Vectastain dab 3 3 diamino benzidine kit

Tumor samples were fixed overnight in 4% PFA/PBS, washed with PBS and a small part was immersed in 30% sucrose/PBS, embedded in OCT (VWR Chemicals) and frozen, while the rest was embedded in paraffin. 4-μm-thick sections of FFPE (Formalin-Fixed, Paraffin-Embedded) tumors were blocked using 1% BSA in TBS containing 0.05% Tween 20 (Sigma) (TTBS) and incubated with anti-Ki67 (Thermofisher Scientific, MA5-14520) or anti-CD31 (Abcam, ab28364) primary antibodies. A biotinylated secondary anti-rabbit antibody (Vector Laboratories) and the Vectastain ABC kit (Vector Laboratories) were used for signal detection and amplification, respectively. Signal development was performed with the Vectastain DAB (3,3-diamino-benzidine) kit (Vector Laboratories) and hematoxylin was used as a counterstain. Images were acquired with an Eclipse E800 microscope (Nikon) equipped with a QImaging Digital Camera. Cryosections were incubated with the anti-CD31 and anti-VCAM1 (Abcam, ab19569) antibodies, followed by the secondary A568-conjugated anti–rabbit antibody (Invitrogen, A110011) and the A488-conjugated anti-rat antibody (Invitrogen, A11006), respectively. Mounting medium containing DAPI (Sigma-Aldrich) was used to stain the nuclei and images were acquired using a Leica TCS SP8X White Light Laser confocal system. Quantifications were performed using ImageJ software analysis